Whole-genome sequencing reveals that variants in the Interleukin 18 Receptor Accessory Protein 3′UTR protect against ALS

The noncoding genome is substantially larger than the protein-coding genome but has been largely unexplored by genetic association studies. Here, we performed region-based rare variant association analysis of >25,000 variants in untranslated regions of 6,139 amyotrophic lateral sclerosis (ALS) whole genomes and the whole genomes of 70,403 non-ALS controls. We identified interleukin-18 receptor accessory protein (IL18RAP) 3′ untranslated region (3′UTR) variants as significantly enriched in non-ALS genomes and associated with a fivefold reduced risk of developing ALS, and this was replicated in an independent cohort. These variants in the IL18RAP 3′UTR reduce mRNA stability and the binding of double-stranded RNA (dsRNA)-binding proteins. Finally, the variants of the IL18RAP 3′UTR confer a survival advantage for motor neurons because they dampen neurotoxicity of human induced pluripotent stem cell (iPSC)-derived microglia bearing an ALS-associated expansion in C9orf72, and this depends on NF-κB signaling. This study reveals genetic variants that protect against ALS by reducing neuroinflammation and emphasizes the importance of noncoding genetic association studies.

The noncoding genome is substantially larger than the protein-coding genome but has been largely unexplored by genetic association studies. Here, we performed region-based rare variant association analysis of >25,000 variants in untranslated regions of 6,139 amyotrophic lateral sclerosis (ALS) whole genomes and the whole genomes of 70,403 non-ALS controls. We identified interleukin-18 receptor accessory protein (IL18RAP) 3′ untranslated region (3′UTR) variants as significantly enriched in non-ALS genomes and associated with a fivefold reduced risk of developing ALS, and this was replicated in an independent cohort. These variants in the IL18RAP 3′UTR reduce mRNA stability and the binding of double-stranded RNA (dsRNA)binding proteins. Finally, the variants of the IL18RAP 3′UTR confer a survival advantage for motor neurons because they dampen neurotoxicity of human induced pluripotent stem cell (iPSC)-derived microglia bearing an ALS-associated expansion in C9orf72, and this depends on NF-κB signaling. This study reveals genetic variants that protect against ALS by reducing neuroinflammation and emphasizes the importance of noncoding genetic association studies.
ALS is a fatal neurodegenerative syndrome that primarily affects the human motor neuron system and has a strong genetic predisposing component 1,2 . Thus far, mutations in approximately 25 protein-coding genes have been associated with ALS 1,3-5 . Hexanucleotide repeat expansion in an intronic sequence of the C9orf72 gene is the most common genetic cause of ALS [6][7][8] , and enrichment of variants was recently discovered in the CAV1 enhancer 9 . However, noncoding nucleotide variants in ALS have yet to be systematically explored.
Gene-based rare variant association analysis is a genetics approach that is based on the rationale that different rare variants in the same gene may have a cumulative contribution 10 . Therefore, rare variant analytical approaches allow for the identification of genes containing an excess of rare and presumably functional variation in affected individuals relative to healthy individuals. Although mutations in noncoding regions are expected to be numerous 11,12 and were recently shown in family-based autism studies 13 , variants in noncoding regions are not routinely included in rare variant association studies. The application of rare variant association analysis to noncoding regulatory regions is constrained by the availability of whole-genome sequencing (WGS) data and the ability to recognize functional variants in noncoding regulatory regions, which is currently far less effective than for protein-coding genes.

Rare variants in the IL18RAP 3′UTR are associated with ALS
To test whether genetic variations in noncoding regulatory regions are associated with ALS, we analyzed regions of interest in WGS data from the Project MinE ALS sequencing consortium 54 (Extended Data Fig. 1a,b and Supplementary Tables 1 and 2). The discovery cohort consisted of 3,955 individuals with ALS and 1,819 age-and sex-matched healthy individuals, for a total of 5,774 whole genomes from the Netherlands, Belgium, Ireland, Spain, United Kingdom, United States and Turkey (Project MinE Datafreeze 1). We performed a region-based rare variant association test 55 , in which rare genetic variants with minor allele frequencies (MAF) ≤ 0.01 from genomic regions of interest, were binned together to weight their contribution to disease; Region-based rare variant association test was performed on 295 noncoding 3′UTRs of candidate genes that were linked to sporadic ALS via genome-wide association studies 56 or genes encoding RNA-binding proteins (Supplementary Table 3). In addition, we tested all autosomal human pre-miRNA genes (1,750 pre-miRNAs; miRBase v20 (ref. 57 )).
As a positive control, we also performed an association analysis of rare variants in the open reading frames of these 295 genes. For the proteins, we called variants that are predicted to cause frameshifting, alternative splicing, an abnormal stop codon or a deleterious nonsynonymous amino acid substitution that was detected in ≥three of seven independent dbNSFP prediction algorithms 58 (Fig. 1a and Supplementary Table 3). In total, 30,721 rare qualifying variants were identified (Supplementary Table 4). Optimized sequence kernel association test (SKAT-O) 55 identified a significant excess of deleterious minor alleles in the ALS genes NEK1 (127 individuals with ALS and 19 healthy individuals (3.21% and 1.04%): P = 8 × 10 −7 ; P corrected = 2.3 × 10 −4 ), comparable with a reported prevalence of 3% (ref. 59 ), and SOD1 (36 individuals with ALS (0.91%) and 0 healthy individuals: P = 2.6 × 10 −4 and P corrected = 3.73 × 10 −2 ) 60 , which is below the reported 2% prevalence 3 Table 5). Other known ALS genes did not reach statistical significance (Supplementary Table 3), consistent with reported statistical power limitations of Project MinE WGS data in assessing the burden of rare variants 62 . Our analysis did not consider the C9orf72 hexanucleotide (GGGGCC) repeat expansion region.
The rare variant association test did not identify a disease association for any of the autosomal pre-miRNAs in the human genome, nor for any of the predicted genetic networks based on variants aggregated over specific mature miRNAs and their cognate downstream  Fig. 2b,c).
When we tested the association of rare variants in 3′UTRs, the strongest association found was for the 3′UTR of IL18RAP (also known as AcPL/CD218b/IL-18R-β; Fig. 1b, Extended Data Fig. 2d and Supplementary Table 5). This association was higher than expected at random (P = 1.93 × 10 −5 ; P corrected = 5.41 × 10 −3 ) and from the association gained for all protein-coding ALS genes in this cohort, with the exception of NEK1. Notably, the signal was more prevalent in healthy individuals (12/1,819, 0.66%) than in individuals with ALS (6/3,955, 0.15%), indicating that these variants might act as protective variants against ALS.
The IL18RAP 3′UTR was also ranked as the top hit by three other algorithms: the sequence kernel association test (SKAT; P = 1.77 × 10 −5 ; permutated P < 10 −4 ), the combined multivariate and collapsing (CMC) analysis (P = 8.78 × 10 −4 ) or variable threshold (VT) with permutation analysis (permutated P = 1.75 × 10 −3 ), suggesting that the association does not depend on a particular statistical genetics method (Extended Data Fig. 3a-c). Furthermore, when we tested the association of rare variants in miRNA recognition elements in 3′UTRs (variants that are potentially either abrogating conserved miRNA-binding sites or creating new miRNA-binding sites in 3′UTRs), the strongest association was also gained for the 3′UTR of IL18RAP (SKAT-O, P = 3.42 × 10 −5 ; Extended Data Fig. 3d  Together, IL18RAP 3′UTR sequence variants are associated with a nearly fivefold lower risk of suffering from ALS, although it did not reach conventional exome-wide multiplicityadjusted significance threshold (α ≈ 2.6 × 10 −6 (ref. 10 )) in our study.
To investigate the source of the signal in the IL18RAP 3′UTR in a post hoc analysis, we divided the 11 variant nucleotides into two subsets of either 9 singleton variants (9 variants/3 healthy individuals/6 individuals with ALS) or 2 variants that were identified solely in healthy individuals (2 variants/9 healthy individuals/0 individuals with ALS  (Fig. 3a,b). In addition, LCLs from both healthy individuals and individuals with ALS harboring IL18RAP 3′UTR variants significantly down-regulated IL18RAP mRNA and protein expression (Fig. 3a,b). Phosphorylation of NF-κB (p-NF-κB), an established intracellular effector downstream of IL-18 signaling, was similarly higher in the ALS LCLs with canonical IL18RAP 3′UTR and also significantly reduced in control and ALS LCLs, harboring IL18RAP variants (Fig. 3c,d). Consistent results were obtained with C9orf72 hexanucleotide expansion ALS LCLs (Extended Data Fig. 5). Accordingly, variants of the IL18RAP 3′UTR reduced NF-κB activity relative to the canonical 3′UTR in an NF-κB reporter assay in U2OS cells (Extended Data Fig. 6). Therefore, variant forms of the IL18RAP 3′UTR correlate with reduced expression of endogenous IL18RAP and reduced NF-κB signaling.

Variant 3′UTR destabilizes IL18RAP mRNA in human microglia
To further establish the functional relevance of the IL18RAP 3′UTR variants, we edited the genome of human iPSCs donated by individuals with ALS with a C9orf72 repeat expansion 65  We explored the receptive cell type involved in IL-18R signaling by profiling dissociated mouse brain cells, namely, neurons, microglia and astrocytes. Fluorescence cytometric gating on CD11b + and CD45 + and IL18RAP (CD218b) revealed that IL18RAP is mainly expressed on microglia cells (Extended Data Fig. 7a-c). Although IL-18 and IL18RAP expression increases in ALS motor neurons (Extended Data Fig. 8a-c), our observations are consistent with the accepted notion that the role of IL-18 and other cytokines in disease heavily rests on a chronic inflammatory state established particularly by microglia 66 .
Therefore, we next differentiated the isogenic IL18RAP 3′UTR lines into human microglia following the protocol of Haenseler et al. 67 (Fig. 4a). iPSC-derived microglia differentiation was validated by immunofluorescence staining of the microglial-specific marker TMEM119 (Extended Data Fig. 9). In differentiated human microglia, we detected a ~five-to sixfold downregulation in the levels of the variant IL18RAP protein and in the levels of the IL18RAP mRNA relative to the canonical sequence of the isogenic line (Fig. 4b,c).
Therefore, the variants at the 3′UTR regulate IL18RAP mRNA and protein expression and provide a conceivable explanation for the variant function in human C9-ALS microglia. Next, we investigated the molecular mechanism that controls the IL18RAP mRNA levels by performing an mRNA stability assay in human microglia. We measured an mRNA degradation rate that is twice as fast with the rare 3′UTR variants relative to the canonical sequence, after inhibition of mRNA transcription by actinomycin D (Fig. 4d). Thus, the mechanism for reduced IL18RAP mRNA levels is associated with the destabilization of IL18RAP mRNA via variants in the 3′UTR.

Variant 3′UTR reduces binding of dsRNA-binding proteins
We sought the potential trans-acting factors that might differentially bind to the canonical and variant 3′UTRs. To this end, we performed RNA pulldown assays and mass spectrometry on in vitro transcribed canonical and variant forms of the IL18RAP 3′UTRs V1 and V3 (Fig. 5a). Mass spectrometry after pulldown identified 552 proteins with good confidence (passed all quality control filters, found in 50% of the repeats in at least one experimental group and were represented by at least two unique peptides; Supplementary  10a).
In accordance, RNA Fold analysis predicted that the canonical 3′UTR sequence consists of a more stable dsRNA structure than the V1 variant sequence (minimum free energy of canonical and variant IL18RAP 3′UTRs, -39.9 kcal mol −1 and -27.8 kcal mol −1 , respectively; Fig. 5f and Extended Data Fig. 10b). In light of these results, we propose that variant-dependent changes to the secondary structure of the IL18RAP 3′UTR attenuate the binding of one or more of the dsRNA-binding proteins and may be involved in controlling the stability of IL18RAp mRNA.

Variant IL18RAP 3′UTR reduces microglia neurotoxicity
To study the potential protective impact of IL18RAP 3′UTR mutations, we performed survival analyses in a coculture system of human iPSC-derived isogenic IL18RAP 3′UTR microglia (on a C9orf72 repeat expansion background) with human iPSC-derived lower motor neurons (i 3 LMNs; healthy, non-ALS 75 ). Time-lapse microscopy was used to quantify motor neuron survival after microglia activation with a cocktail of lipopolysaccharide (LPS) and the cytokine IL-18 ( Fig. 6a). Motor neuron survival was significantly improved in the presence of microglia harboring the IL18RAP 3′UTR variants relative to microglia harboring the canonical IL18RAP 3′UTR (two independent isogenic pairs based on independent human C9orf72 lines; n = 3 independent differentiation procedures from different passages per line with three to eight coculture wells per passage; Fig. 6b-d and Supplementary Video 1). Based on these studies, we conclude that rare variants of the IL18RAP 3′UTR increase C9orf72 microglia-dependent motor neuron survival and hence convey a protective property.

Variant IL18RAP 3′UTR endows survival advantage to individuals with ALS
To determine whether the variants in the IL18RAP 3′UTR are also protective in individuals with ALS, we tested the association between age of diagnosis and age of death in individuals with ALS harboring canonical or variant forms of IL18RAP 3′UTR. Of 4,216 individuals for whom data on the age of diagnosis were available (Project MinE and NYGC cohorts), 8 harbored IL18RAP 3′UTR variants. Of 4,263 individuals for whom the age of death was available, 9 harbored IL18RAP 3′UTR variants. IL18RAP 3′UTR variants are expected to be depleted in ALS genomes; nonetheless, in those extremely rare individuals harboring IL18RAP 3′UTR variants, these were associated with an older age of death and an older age of diagnosis. On average, the age of death was higher by 6.1 years than that observed for individuals with canonical IL18RAP 3′UTR (permutation P = 0.02; Cohen's d effect size = 0.65; Fig. 6e and Supplementary Table 11), and the age of diagnosis was higher by 6.2 years than that observed for individuals with canonical IL18RAP 3′UTR (permutation P = 0.05; Cohen's d effect size = 0.62; Fig. 6f and Supplementary Table 11). Thus, variants in IL18RAP 3′UTR are protective against ALS in a tissue culture model and correlate with survival advantage for individuals suffering from the disease.

Variant IL18RAP 3′UTR dampens NF-κB signaling in microglia
To study the role of NF-κB signaling in our system, we analyzed NF-κB phosphorylation and the impact on the transcriptome after microglia activation (Fig. 7a). Western blot analysis revealed reduced levels of p-NF-κB in variant IL18RAP 3′UTR relative to isogenic control ( Fig. 7b). Reduced phosphorylation is associated with decreased nuclear localization and transcriptional activity of NF-κB [76][77][78][79] . In parallel, we conducted a next-generation sequencing study (Supplementary Table 12 Table 13). In addition, an unsupervised study of NF-κB pathway mRNAs (GO:0007249) demonstrated broad downregulation of pathwayassociated mRNAs in microglia with the variant IL18RAP 3′UTR relative to the isogenic control ( Fig. 7e). Therefore, the microglial NF-κB transcriptomic signature depends on signaling via the IL-18 receptor and is attenuated by protective IL18RAP 3′UTR variants.
To test a plausible neurotoxic role for NF-κB downstream of the IL-18R in this system, we next performed a coculture survival assay with or without IKK16, a selective IκB kinase (IKK) inhibitor that inhibits NF-κB signaling 80 . In human microglia with the canonical IL18RAP 3′UTR, IKK16 significantly ameliorated motor neuron toxicity relative to control (carrier alone; Fig. 7f). However, in human microglia with the protective variant IL18RAP 3′UTR, inhibition of NF-κB had no effect (two independent isogenic pairs based on independent human C9orf72 lines with three to eight coculture wells per line; Fig. 7f). This suggests that NF-κB's neurotoxic function resides epistatically downstream of IL18RAP in human microglia. Together, rare variants in the IL18RAP 3′UTR diminish NF-κB signaling, thus increasing C9orf72 microglia-dependent motor neuron survival.

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Discussion
By performing rare variant aggregation analysis in regulatory noncoding regions on data from Project MinE and NYGC ALS sequencing consortia, we demonstrate that variants in the 3′UTR of IL18RAP are enriched in non-ALS genomes, indicating that these are relatively depleted in ALS. IL18RAP 3′UTR variants reduced the chance of developing ALS fivefold and delayed onset and therefore age of death in individuals with ALS.
We demonstrate the downregulation of variant IL18RAP 3′UTR, which might be particularly relevant because elevated levels of the cytokine IL-18 (refs. [39][40][41] ) and IL18RAP were measured in several forms of ALS. Mechanistically, we demonstrate destabilization of IL18RAP by the variant 3′UTR, which is consistent with the reduced propensity of the 3′UTR to form a double-stranded secondary structure and accordingly, reduced binding of dsRNA-binding proteins that are known to stabilize mRNAs. As a consequence, IL18RAP expression and NF-κB signaling are dampened in microglia.
We demonstrate the neuroprotective effect of the variant IL18RAP 3′UTR using CRISPRedited human isogenic C9orf72 microglia. Mechanistically, it is because IL18RAP is epistatically upstream of NF-κB in this system. Thus, the variant IL18RAP 3′UTR attenuates NF-κB activity and the expression of a broad set of NF-κB effector genes.
Our study resonates with the existence of protective variants in protein-coding regions in Alzheimer's disease [81][82][83][84] and in ALS 85,86 and emphasizes the importance of seeking functional protective variants in association studies in neurodegeneration.
The discovery of a protective non-protein-coding allele is in line with previous reports of non-protein-coding variants in VEGF promoter/5′UTR, pre-miR-218-2 and CAV1/CAV2 enhancers 9,87,88 , which were all deleterious. Together, these studies underscore the need to systematically explore the noncoding genome in brain diseases.
One limitation of our study is that the IL18RAP 3′UTR signal did not reach the conventional exome-wide multiplicity-adjusted significance threshold (α ≈ 2.6 × 10 −6 ; ref. 10 ). However, the IL18RAP 3′UTR signal is comparable to that of protein-coding ALScausing genes, such as SOD1 and NEK1. Furthermore, the key findings were reproduced in a genome-wide study of all human 3′UTRs and in an independent replication study. Limitations in the statistical power might have prevented the discovery of other noncoding variants and may be overcome with larger ALS and control cohorts, which are not currently available. Additionally, we have focused our tissue culture studies on human C9orf72 microglia. Therefore, the involvement of the IL18RAP 3′UTR in other ALS-associated genetic backgrounds remains to be experimentally explored, as is the relevance to other neurodegenerative diseases. Finally, the mechanism underlying IL18RAP dose sensitivity is not fully understood. While we provide evidence that the variant IL18RAP 3′UTR endows neuroprotection via dampening of microglia-dependent neurotoxicity, additional In general, sample size was measured based on collecting as many available ALS genomes and matched healthy controls. No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications 9, 62 . The human genetics study was a case-control cohort study; therefore, randomization of experimental groups and blinding was not relevant. All analyses for molecular biology studies were performed in a blinded manner.

Quality control procedures in Project MinE genomics
Sample selection, data merging and sample-and variant-level quality control procedures for Project MinE ALS sequencing consortium genomes are described in full previously 62 . Briefly, 6,579 Project MinE ALS sequencing consortium whole genomes were sequenced on Illumina HiSeq2000 or HiSeqX platforms. Reads were aligned to the human genome build hg19, and sequence variants were called with the Isaac pipeline 92 . Individual genomic variant call format files (GVCFs) were merged with the 'agg' tool, a utility for aggregating Illumina-style GVCFs. Following completion of the raw data merge, multiple quality control filtering steps were performed: (1) setting genotypes with GQ < 10 to missing, (2) removing low-quality sites (QUAL < 30 and QUAL < 20 for SNPs and indels, respectively), (3) removing sites with missingness of > 10%, (4) samples were excluded if they deviated from the mean by more than 6 s.d. for total numbers of SNPs, singletons and indels, transition/ transversion (Ti/Tv) ratio, heterozygous/homozygous-non-reference ratio and inbreeding (by cohort), (5) missingness > 5%, (6)  individuals with ALS and 1,819 healthy individuals) passed all quality control measures and were included in downstream analysis. Per-nucleotide site quality control was performed on quality control-passing samples only; for biallelic sites: variants were excluded from analysis based on total depth (DP < 10,000 or >226,000), missingness of >5%, passing rate in the whole data set of <70%, sites out of Hardy-Weinberg equilibrium (by cohort, controls only, P < 1 × 10 −6 ) and sites with extreme differential missingness between samples from individuals with ALS and healthy individuals (overall and by cohort, P < 1 × 10 −6 ).
Non-autosomal chromosomes and multiallelic variants were excluded from analysis.

Selection of regions of interest
Discontinuous regions of interest approximating in total ~5 Mb include coding sequences and 3′UTRs of 295 genes (Supplementary Table 3) encoding proteins that were (1) previously reported to be associated with ALS, (2) RNA-binding proteins, including miRNA biogenesis or activity factors (University of California Santa Cruz gene annotation 93

Annotation and region-based rare variant association analysis
After quality control and extraction of regions of interest, we performed functional annotation of all variants. Indels were left aligned and normalized using bcftools, and multiallelic sites were removed. For variant annotation, we developed a pipeline that calculates the impact of genetic variation in coding regions and in 3′UTR and miRNA regions using ANNOVAR 95  Noncoding sequence region-based rare variant association analysis included 3′UTRs and variants in miRNA recognition elements in 3′UTRs (Supplementary Table 3). Variants that impaired conserved miRNA-binding sites in 3′UTRs (predicted loss of function) were called by TargetScan (v7.0) 96 . Newly created miRNA-binding sites in 3′UTRs (predicted gain of function) were called by textual comparison of all possible mutated seeds around a variant to all known miRNA seed sequences in the genome, all human pre-miRNAs (mirBase v20) 57 and miRNA:target gene networks, mature miRNA sequences (mirBase v20) 57 and cognate targets within the 3′UTRs (Supplementary Table 3). Variant annotation scripts are available at GitHub at https://github.com/TsviyaOlender/Non-coding-Variants-in-ALS-genes-.

Isolation and culture of rat cortical astrocytes
All experiments were performed in accordance with relevant guidelines and regulations of the Institutional Animal Care and Use Committee at Weizmann Institute of Science (IACUC 09491120-1). Primary cortical astrocytes were isolated and cultured as previously described 97 with several modifications. Briefly, the cerebral cortex of postnatal day 1 male Sprague-Dawley rat pups (purchased from Envigo) was dissected and placed in DMEM/F12 containing 0.5% trypsin (Biological Industries, 03-046-5B). After a 30-min incubation at 37 °C in a water bath, the cortical tissues were mechanically dissociated with a pipette into single cells and were seeded on poly-D-lysine-coated (Sigma-Aldrich, 7405) T75 culture flasks in astrocyte medium (DMEM/F12 (Gibco, 31330) supplemented with 10% FBS, 50 U ml −1 penicillin-streptomycin and 2 mM GlutaMAX (Gibco, 35050038)). The confluent cultures were shaken for 4 h at 200 r.p.m. to remove microglial cells. Each T75 flask was trypsinized and split into three new T75 flasks. After 7-8 d, the confluent flasks were trypsinized and frozen in 90% FBS and 10% DMSO until further use.

Generation of IL18RAP 3′UTR rare variant human iPSC lines
iPSCs were generated by the Ichida lab from human lymphocytes from individuals with ALS obtained from the NINDS Biorepository at the Coriell Institute for Medical Research.
Lymphocytes were reprogrammed into iPSCs as previously described 65  CRISPR guides were chosen using several design tools, including the MIT CRISPR design tool 98 and sgRNA Designer, Rule set 2 (ref. 99 ), in the Benchling implementations (www.benchling.com) and SSC 100 and sgRNAscorer 101 in their websites.
Before the CRISPR procedure, iPSCs were passaged once under feeder-free conditions (LDEV Free GelTrex matrix (Gibco, A1413202) and mTESR1 medium (StemCell Technologies, 85850)), dislodged as single cells using StemPro Accutase (Gibco,  A11105- Table 15; IDT, 400 bases Megamer DNA Oligonucleotide) and 2.6 μg of carrier plasmid DNA. CRISPR reaction components were introduced to iPSCs by singleround electroporation using a Nepa21 system (NEPA GENE). One hundred microliters of cells and DNA suspension was transferred to a Nepa Electroporation Cuvette, 2-mm gap (Nepa Gene, EC-002). The following electroporation conditions were used: 150-V poring pulse, 5-ms pulse length, 20-V transfer pulse and 50-ms pulse length. Electroporated cells were transferred to two GelTrex-coated 100-mm dishes (1,000 and 10,000) in mTeSR medium supplemented with 10 μM ROCK inhibitor (PeproTech, 1293823) and placed into a CO 2 incubator for 2 d. Forty-eight hours after electroporation, cells were treated with 0.5 μg ml −1 puromycin (Sigma-Aldrich) for 2 consecutive days. Cells that survived were maintained until clone development. Single clones were picked and transferred to 96-well plates. Matured clones were genotyped at the first passage. Additionally, the top five predicted off-target sites for the gRNA were sequenced (Supplementary Table 16). Selected clones containing desired mutations were expanded, cryopreserved and used for the downstream experiments.

Differentiation and culturing of human iPSC-derived microglia
Human iPSCs were differentiated into microglia-like cells as previously described 67 Table 18) overnight at 4 °C with rocking in antibody solution (5% albumin, 0.02% sodium azide and five drops of phenol red in 0.05% PBST). Following primary antibody incubation, membranes were washed three times for 5 min at room temperature with 0.05% PBST and incubated for 1 h at room temperature with horseradish peroxidase-conjugated species-specific secondary antibodies, washed three times for 5 min each in 0.05% PBST at room temperature and visualized using EZ-ECL Chemiluminescence (Biological Industries, 20500-120) by ImageQuant LAS 4000 (GE Healthcare Life Sciences). Densitometric analysis was performed using ImageJ v ij-1.52n (NIH).

In vitro transcription of biotinylated IL18RAP 3′UTR
To identify the potential trans-acting factors that might differentially bind to the canonical and variant 3′UTRs, an RNA pulldown and mass spectrometry assay was performed on in vitro transcribed canonical and variant forms of the IL18RAP 3′UTRs, V1 and V3. Briefly, The canonical, V1 and V3 biotinylated IL18RAP 3′UTR sequences (384 nucleotides) and the negative control (ultrapure water only) were produced by using an in vitro transcription HiScribe T7 ARCA kit (NEB, E2060S) following the manufacturer's instructions. Briefly, 300 ng of purchased DNA template (50 ng μl −1 ; Twist; Supplementary

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Europe PMC Funders Author Manuscripts (wild-type, V1, V3 and negative control; n = 6 repeats per group) to the beads, new prepared binding Pierce streptavidin magnetic beads were incubated by rotation with equal amounts of in vitro transcribed products for 30 min at 4 °C (100 μl of beads per 10 pmol of RNA product). After 30 min, the tubes of incubated in vitro transcribed products with beads were washed three times, and the cleared lysate was added equally to each tube and incubated for 30 min at 4 °C. In the next step, the samples were washed three times by magnetizing the beads and resuspended by vortexing with a high-salt buffer. The bound beads were magnetized and suspended in 20 μl of RNase-free 1× PBS for the on-bead digestion procedure.

Liquid chromatography and mass spectrometry
The resulting peptides were analyzed using nanoflow liquid chromatography (nanoAcquity) coupled to high-resolution, high mass accuracy mass spectrometry (Q-Exactive HF). Each sample was analyzed on the instrument separately in a random order in discovery mode.

Raw proteomic data processing
Raw mass spectrometry data were processed using MaxQuant version 1.6.6.0 (ref. 107 ). A database search was performed with the Andromeda search engine 108,109 using the human UniProt database appended with common lab protein contaminants. A forward/ decoy approach was used to determine the FDR and filter the data with a threshold of 1% FDR for both the peptide-spectrum matches and the protein levels. The label-free quantification algorithm in MaxQuant 110 was used to compare between experimental samples. Additional settings included the following modifications: fixed modification, cysteine carbamidomethylation; variable modifications, methionine oxidation, asparagine and glutamine deamidation and protein N-terminal acetylation.

Proteomics statistical analysis
The ProteinGroups output table was imported from MaxQuant to the Perseus v.1.6.2.3 environment 111 . Quality control excluded reverse proteins, proteins identified only based on a modified peptide and contaminants. Non-specific streptavidin bead binders were excluded by the following procedure: label-free quantification intensity values were log 2 transformed, and two outlier samples were excluded from further analysis. Missing values were imputed by creating an artificial normal distribution with a downshift of 1.8 s.d. and a width of 0.4 of the original ratio distributions. A Student's t-test with s0 = 0.1 was performed with an FDR P value of ≤0.05 between the experimental groups (canonical, V1 and V3) and the negative control group, which was defined as a single control group. Proteins that passed all quality control filters were separated for each of the experimental groups and compared to the negative control samples (ultrapure water). The statistically significantly associated proteins were filtered to retain only proteins that were found in 50% of the repeats in at least one experimental group and were represented by at least one unique peptide. The enriched proteins were subjected to Student's t-test between every two groups (canonical versus V1 and canonical versus V3), with S 0 = 0.1, FDR P ≤ 0.05 and a fold change threshold of >2.

Processing of mouse brain samples for flow cytometry
Male C57BL/6J wild-type mice (~130 d old; n = 5 per experiment) were used for all mouse experiments unless otherwise specified. Mice were housed in groups, with a maximum of six mice per cage, in a passive air flow, environmentally ventilated cage system and maintained under specific pathogen-free conditions at the Walter and Eliza Hall Institute Animal Facility. Mouse cages were housed in an environmentally controlled room that was maintained at 20-21 °C with 60% relative humidity and a 14-h light/10-h dark cycle (6:00-20:00) with no external or natural light sources. Mice were killed with CO 2 and perfused with PBS through the left ventricle of the heart. Dissected mouse cortex was cut into smaller pieces using scissors and digested in 0.5 mg ml −1 collagenase IV (Worthington Biochemical), 10 μg of deoxyribonuclease (Sigma-Aldrich) and 10% HI-FBS, RPMI1640 (Gibco) at 37 °C for 30 min with continuous agitation. Digested samples were gently triturated for 1 min, and the enzymatic reaction was stopped by adding 1 mM EDTA in PBS. The homogenate was filtered through a 100-μm cell strainer and centrifuged at 400g for 8 min at 4 °C to pellet the cells and myelin. This was followed by a myelin removal step by gradient centrifugation with 30% Percoll (Sigma-Aldrich) in PBS (700g for 20 min at 21 °C without brakes during deceleration). After myelin (the top white layer) separation, the middle transparent layer was collected, washed in PBS and centrifuged at 400g for 8 min at 4 °C to pellet the cells. Data were collected as FCS files and analyzed with FlowJo v10 software (BD Biosciences). Antibody specificity was assessed using relevant isotype control antibodies and fluorescence minus one. Compensation was adjusted using single-stained samples.
The expression of IL18RAP (IL-18Rβ) was expressed as mean fluorescence intensity or percent frequency after gating for the following cell types: immune cells (CD45 hi ), microglia (CD45 int CD11 hi ), neurons (CD45 − CD11b − NeuN + ) and astrocytes (CD45 − CD11b − GFAP + ). Experimental use was in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (Australian National Health

Reporting Summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Extended Data
Extended Data Fig. 1 Table 3. These ORFs encode for ALS-relevant proteins or proteins that are associated with miRNA biogenesis or activity. Variants were depicted if predicted to cause frameshifting, alternative splicing, abnormal stop codon or a deleterious non-synonymous amino acid substitution, in ≥ 3 of 7 independent dbNSFP prediction algorithms (genomic inflation λ = 0.96), (b) All     Table 6).

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.         independent experiments). c-e, mRNA extracted from human microglia was subjected to a next-generation sequencing with downstream bioinformatics studies. ORA within KEGG Pathways (c) and GO biological processes (d) of the differentially expressed transcriptome in microglia harboring variant versus canonical IL18RAP 3′UTR. Bar graphs depicting the ratio of enrichment for significantly enriched pathways (FDR ≤ 0.05) are shown (Supplementary Table 13; WebGestalt 89 ). An unsupervised study of the NF-κB transcriptomic signature (GO:0007249 pathway-associated mRNAs) in microglia with the variant (blue) relative to the isogenic canonical (gray) IL18RAP 3′UTR is shown (e). High expression is in red, and low expression is in blue. f, Time-lapse microscopic analyses of cocultured human i 3 LMNs (healthy, non-ALS 75 ) with human iPSC-derived isogenic IL18RAP 3′UTR microglia (on a C9orf72 repeat expansion background), activated with a cocktail of LPS and the cytokine IL-18, without (carrier alone; DMSO) or with IKK16 (200 nM), a selective IKK inhibitor that inhibits NF-κB signaling 80 . IKK16 significantly ameliorates motor neuron death relative to control only in the context of canonical IL18RAP 3′UTR, but did not further contribute to rescue in human microglia with the protective variant IL18RAP 3′UTR; two independent isogenic pairs based on independent human C9orf72 lines with three to eight coculture wells per line (a survival plot with mean and s.e.m. values is shown; two-way ANOVA; C9-ALS microglia line 1, P = 0.01; C9-ALS microglia line 2, P = 0.025; *P < 0.05).